SUMMARY About 10-20% of Americans age 65+ have Mild Cognitive Impairment (MCI), of which ~15% progress to dementia annually. A growing number of companies are developing new therapeutics aiming to intervene early in the development of Alzheimer's disease, the most common form of dementia, and there is a great need for accurate, minimally invasive diagnostics for primary screening of individuals with early, including presymptomatic, stages of the disease. DiamiR has developed a platform technology for early detection and differential diagnosis of AD and other neurodegenerative diseases (NDs) based on targeted selection and analysis of brain-enriched and inflammation-associated microRNAs (miRNAs) circulating in plasma. A fundamental hypothesis underlying our approach is that brain-enriched miRNAs, which are present in neurites and synapses, enriched in different brain regions affected by pathology (e.g. hippocampus for early AD, cortex and midbrain for advanced AD), and detectable in plasma present effective biomarker candidates for detecting AD at different stages. Normalization by other brain-enriched miRNAs is used to compensate for changes unrelated or non-specific to the pathology. Since the development of NDs is often accompanied by neuroinflammation, certain inflammation-associated miRNAs were also included in the analysis. In the Preliminary, SBIR Phase I and II studies conducted in collaboration with the Roskamp Institute, Washington University and University of Pennsylvania, miRNA pairs capable of detecting MCI and AD at different stages of disease development, including presymptomatic stage, and differentiating AD from Parkinson's disease, frontotemporal lobar degeneration, and amyotrophic lateral sclerosis with over 80% accuracy have been established and tested in multiple cohorts. The goal of this SBIR Phase IIB study is to optimize the protocol for sample preparation and processing so as to minimize miRNA degradation and maximize data reproducibility, and to develop and validate Clinical Trial Assays (CTAs) for MCI and AD detection. Specific aims include (1) optimization of the protocol for sample preparation; collection of plasma samples using the optimized protocol from MCI, AD, control subjects, feature reduction to retain 12-16 miRNAs; (2) development, analytical validation of CTAs; and (3) assessment of the assay performance for the detection of MCI, AD, presymptomatic AD (cognitively normal, A?-positive) and control (cognitively normal, A?-negative) using samples prospectively collected with the optimized protocol at NYU, UPenn, UCSD AD centers; and assessment of the assay performance for the detection of presymptomatic AD using pre-collected plasma samples from the screening for A4 study. The CTAs will be developed under CLIA guidelines and used to facilitate screening of better-defined participants into clinical trials and monitoring of disease progression and response to treatment. Following extensive testing in clinical trial setting, the assays, comprising the diagnostic product CogniMIRTM, will be developed as in vitro diagnostics (IVD) and submitted for the approval by the FDA.